Recovery of isoflavones from soy molasses

ABSTRACT

Methods for recovering isoflavones and derivatives thereof from soy molasses are disclosed. In a first embodiment, a method is disclosed in which isoflavones are recovered without any significant conversion of isoflavone conjugates to other forms. In a second embodiment, a method is disclosed whereby isoflavone conjugates are converted to glucosides while in the soy material prior to their recovery. In a third embodiment, a method is disclosed in which isoflavones are converted to their aglucone form while in the soy material and prior to their recovery. Also disclosed are various isoflavone enriched products obtained from soy molasses.

FIELD OF THE INVENTION

The present invention relates to processes for recovering isoflavonesfrom soy molasses. In addition, the present invention relates to theresulting products containing isoflavones.

BACKGROUND OF THE INVENTION

Isoflavones occur in a variety of leguminous plants, including vegetableprotein materials such as soybeans. These compounds include daidzin,6"-OAc daidzin, 6"-OMal daidzin, daidzein, genistin, 6"-OAc genistin,6"-OMal genistin, genistein, glycitin, 6"-OAc-glycitin, 6"-4OMalglycitin, glycitein, biochanin A, formononentin, and coumestral.Typically these compounds are associated with the inherent, bitterflavor of soybeans.

The isoflavones in soybean materials include isoflavone glucosides(glucones), isoflavone conjugates and aglucone isoflavones. Isoflavoneglucosides have a glucose molecule attached to an isoflavone moiety.Isoflavone conjugates have additional moieties attached to the glucosemolecule of an isoflavone glucoside, for example, 6"-OAc genistincontains an acetate group attached to the six position of the glucosemolecule of genistin. Aglucone isoflavones consist solely of anisoflavone moiety.

Soy contains three "families" of isoflavone compounds havingcorresponding glucoside, conjugate, and aglucone members: the genisteinfamily, the daidzein family, and the glycitein family. The genisteinfamily includes the glucoside genistin; the conjugates 6"-OMal genistin(6"-malonate ester of genistin) and 6"-OAc genistin (6"-acetate ester ofgenistin); and the aglucone genistein. The daidzein family includes theglucoside daidzin; the conjugates 6"-OMal daidzin and 6"-OAc daidzin;and the aglucone daidzein. The glycitein family includes the glucosideglycitin; the conjugate 6"-OMal glycitin; and the aglucone glycitein.

In the production of commercial products, such as vegetable proteinconcentrates, the focus has been to remove these materials. For example,in a conventional process for the production of a soy proteinconcentrate in which soy flakes are extracted with an aqueous acid or anaqueous alcohol to remove water soluble materials from the soy flakes,much of the isoflavones are solubilized in the extract. The extract ofwater soluble materials, including the isoflavones, is soy molasses. Thesoy molasses is a byproduct material in the production of soy proteinconcentrate which is typically discarded. Soy molasses, therefore, is aninexpensive and desirable source of isoflavones, provided that theisoflavones can be separated from the soy molasses.

It has recently been recognized that the isoflavones contained invegetable protein materials such as soybeans have medicinal value. Theaglucone isoflavones are of particular interest. Genistein and daidzeinmay significantly reduce cardiovascular risk factors. "Plant andMammalian Estrogen Effects on Plasma Lipids of Female Monkeys",Circulation, vol. 90,p. 1259 (October 1994). Genistein and daidzein arealso thought to reduce the symptoms of conditions caused by reduced oraltered levels of endogenous estrogen in women, such as menopause orpremenstrual syndrome. Further, it has recently been recognized thataglucone isoflavones may inhibit the growth of human cancer cells, suchas breast cancer cells and prostate cancer cells, as described in thefollowing articles: "Genistein Inhibition of the Growth of Human BreastCancer Cells, Independence from Estrogen Receptors and the Multi-DrugResistance Gene" by Peterson and Barnes, Biochemical and BiophysicalResearch. Communications, Vol. 179, No.1, pp.661-667, Aug. 30, 1991;"Genistein and Biochanin A Inhibit the Growth of Human Prostrate CancerCells but not Epidermal Growth Factor Receptor TyrosineAutophosphorylation" by Peterson and Barnes, The Prostate, Vol.22,pp.335-345 (1993); and "Soybeans Inhibit Mammary Tumors in Models ofBreast Cancer" by Barnes, et al., Mutagens and Carcinogens in the Diet,pp.239-253 (1990).

The aglucone isoflavones have the following general formula: ##STR1##wherein, R₁, R₂, R₃ and R₄ may be selected from the group consisting ofH, OH and OCH₃. Genistein has the formula above where R₁ ═OH, R₂ ═H, R₃═OH, and R₄ ═OH, daidzein has the formula above where R₁ ═OH, R₂ ═H, R₃═H, and R₄ ═OH, and glycitein has the formula above where R₁ ═OH, R₂═OCH₃, R₃ ═H, and R₄ ═OH.

It is therefore to the isoflavones and to the recovery of an isoflavoneenriched material from soy molasses to which the present invention isdirected. The present invention is further directed to isoflavoneglucosides and aglucone isoflavones - to the conversion of isoflavonesof soy molasses to isoflavone glucosides and aglucone isoflavones, andto the recovery of an isoflavone glucoside enriched material and anaglucone isoflavone enriched material from soy molasses.

A general process for converting vegetable protein isoflavone conjugatesto aglucone isoflavones is known, and is provided in the currentlypending application U.S. Ser. No. 08/477,102 filed Jun. 7, 1995 owned bythe assignee of the present application.

Other processes are known in the art for converting isoflavoneglucosides to aglucone isoflavones, such as described in Japanese PatentApplication 258,669 to Obata, et al. Such processes do not provide forthe recovery of an isoflavone enriched material from soy molasses. Suchprocesses also do not provide for the conversion of isoflavoneconjugates to isoflavone glucosides or to aglucone isoflavones.Furthermore, these processes achieve only a moderate extent ofconversion of isoflavone glucosides to aglucone isoflavones, and requirea substantial period of time to effect this moderate extent conversion.

It is therefore an object of the present invention to provide anisoflavone enriched material and a process for producing the same fromsoy molasses.

It is a further object of the present invention to provide an isoflavoneglucoside enriched material and a process for producing the same fromsoy molasses.

It is still a further object of the present invention to provide anaglucone isoflavone enriched material and a process for producing thesame from soy molasses.

SUMMARY OF THE INVENTION

The present invention is an isoflavone enriched material and a processfor recovering the same from a soy molasses material containingisoflavones. The method comprises providing a soy molasses materialcontaining isoflavones, and separating a cake from the soy molassesmaterial at a pH and a temperature sufficient to cause a majority of theisoflavones to be contained in the cake. Preferably the pH is about 3.0to about 6.5 and the temperature is about 0° C. to about 35° C. duringthe separation. The cake is an isoflavone enriched material.

In one embodiment, a glucoside enriched isoflavone material is formedfrom the cake of isoflavone enriched material. An aqueous slurry isformed of the isoflavone enriched material. The slurry is treated at atemperature of about 2° C. to about 1 20° C. and a pH of about 6 toabout 13.5 for a time sufficient to convert isoflavone conjugates in theisoflavone enriched material to isoflavone glucosides. A cake ofisoflavone glucoside enriched material may then be separated from theslurry.

In another embodiment, an aglucone isoflavone enriched material isformed from the cake of isoflavone enriched material. An aqueous slurryis formed of the isoflavone enriched material. The slurry is treated ata temperature of about 2° C. to about 1 20° C. and a pH of about 6 toabout 13.5 for a time sufficient to convert isoflavone conjugates in theisoflavone enriched material to isoflavone glucosides. An enzyme capableof cleaving 1,4-glucoside bonds is contacted with the isoflavoneglucosides in the slurry at a temperature of about 5° C. to about 75° C.and a pH of about 3 to about 9 for a time sufficient to convert theisoflavone glucosides to aglucone isoflavones. A cake of agluconeisoflavone enriched material may be separated from the slurry.

In another aspect, the present invention is an isoflavone glucosideenriched material and a process for recovering the same from soymolasses. The soy molasses is treated at a temperature of from about 2°C. to about 120° C. and at a pH value of between about 6 to about 13.5for a time period sufficient to convert isoflavone conjugates containedin the soy molasses to isoflavone glucosides. A cake of isoflavoneglucoside enriched material is separated from the soy molasses materialat a pH and a temperature sufficient to cause a majority of theisoflavone glucosides to be contained in the cake.

In another aspect, the present invention is an aglucone isoflavoneenriched material, and a process for recovering the same from soymolasses. The soy molasses is treated at a temperature of from about 2°C. to about 120° C. and at a pH value of between about 6 to about 13.5for a time period sufficient to convert isoflavone conjugates containedin the soy molasses to isoflavone glucosides. An enzyme capable ofcleaving 1,4-glucoside bonds is contacted with the isoflavone glucosidesin the soy molasses material at a temperature of about 5° C. to about75° C. and a pH of about 3 to about 9 for a time period sufficient toconvert the isoflavone glucosides to aglucone isoflavones. A cake ofaglucone isoflavone enriched material is separated from the soy molassesmaterial at a pH and a temperature sufficient to cause a majority of theaglucone isoflavones to be contained in the cake.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The starting material for the processes described herein is soymolasses. Soy molasses is a by-product of many commercial processesinvolving soybeans or soybean derivatives, such as processes forproducing protein extracts, protein whey, and protein concentrateproducts. Accordingly, soy molasses is produced in large quantities tosuch an extent that soy molasses is a relatively inexpensive commodity.

Soy molasses is generally considered to be the soy solubles removed fromsoy insolubles by washing with alcohol or acid. Condensed soy molassesis typically an aqueous mixture containing about 50% or more solids thatcomprise about 6% (based on the total weight of the soy molasses)protein, about 3% ash, about 5% fat, and about 36% carbohydrates. Soymolasses is also known in the art as soya bean solubles. "Soy molassesmaterial" as that term is used herein refers to a composition containingsoy molasses, and/or derivatives of soy molasses such as an isoflavoneglucoside enriched soy molasses material and an aglucone isoflavoneenriched soy molasses material. Accordingly, these terms are usedinterchangeably herein.

In a preferred embodiment, the soy molasses starting material isproduced from defatted soy flakes from which oil has been removed bysolvent extraction in a conventional manner. The defatted soy flakes areextracted with water which has been adjusted to an acidic pH, preferablyabout pH 4 to about pH 5, by the addition of one or more suitable acidssuch as acetic acid, sulfuric acid, phosphoric acid, hydrochloric acidor any other suitable reagent. Preferably the ratio of the acidicextractant to soy flakes is about 16:1 to about 20:1 by weight. Toimprove the efficacy of the extraction, the temperature of theextractant may be elevated above room temperature, preferably betweenabout 32° C. and about 55° C. After the extraction, the soy molassesextract is removed from the soy insolubles.

In another embodiment, defatted soy flakes are extracted with aqueousalcohol to produce the soy molasses starting material. Preferably, theflakes are extracted with about 80% aqueous ethanol at a ratio of about16:1 to about 20:1 by weight of extractant to soy flakes. Thetemperature of the alcohol extractant may be elevated above roomtemperature, preferably from about 32° C. to about 55° C., to improvethe efficacy of the extraction. The soy molasses extract is then removedfrom the soy insolubles to provide the soy molasses starting material.

An isoflavone enriched material may be recovered from the soy molassesstarting material. The soy molasses material may be diluted with waterto a solids content of from about 6% to about 13%, with 13% being mostpreferred. Dilution of the soy molasses material is not a requirementfor the process, however, diluting the relatively thick soy molassesmaterial facilitates processing the material.

The soy molasses material, preferably diluted, is treated at a pH and atemperature at which a majority of the isoflavones will separate fromthe soy molasses upon performing a separation procedure. In a preferredembodiment, the soy molasses material is treated at a pH of about 3.0 toabout 6.5 and a temperature of about 0° C. to about 35° C. to maximizethe insolubility of the isoflavones in the soy molasses. Isoflavones maybe separated from the soy molasses at pH values outside the preferredrange and at temperatures above 35° C., however, these conditions areless preferred since less isoflavones are separated from the soymolasses in the separation procedure. The pH of the soy molasses may beadjusted with a suitable conventional acidic or basic reagent, ifnecessary. It is most preferred that the pH of the soy molasses beadjusted to about 4.5. It is also preferred to chill or cool the soymolasses to a temperature of about 0° C. to about 10° C., and mostpreferably to a temperature of about 4° C. to about 7° C.

The soy molasses material is then subjected to a separation procedure toseparate a cake of isoflavone enriched material from the soy molassesmaterial. The separation is performed while the soy molasses material ismaintained under the previously described pH and temperature conditions.

In one embodiment, the cake of isoflavone enriched material is separatedby centrifuging the soy molasses and decanting the supernatant from thecake. Centrifugation is preferably performed at about 3,000 to about10,000 rpm for approximately 30 minutes at about 0° C. to about 10° C.

In another embodiment, the isoflavone enriched cake may be separatedfrom the soy molasses material by filtration. Preferably the filtrationis done at the previously described pH and temperature conditions, mostpreferably at a pH of about 4.5 and a temperature of about 0° C. toabout 1° C.

In another aspect of the present invention, a cake of isoflavoneglucoside enriched material may be recovered from soy molasses. A soymolasses starting material is obtained as described above. Although nota requirement, it is preferred that the soy molasses material be dilutedto a solids content of from about 6% to about 13%, and most preferablyabout 13%, to facilitate processing the material.

A conversion operation is then performed on the soy molasses to convertisoflavone conjugates in the soy molasses to isoflavone glucosides. Asubstantial portion of the isoflavones in the soy molasses areisoflavone conjugates, therefore the conversion substantially increasesthe amount of isoflavone glucosides in the soy molasses material. Theconversion has been found to be dependent on the pH and the temperatureof the soy molasses.

The pH range for conversion of the isoflavone conjugates to isoflavoneglucosides in soy molasses is from about 6 to about 13.5. The pH of thesoy molasses should be adjusted to the desired pH, if necessary, with asuitable base, caustic agent, or basic reagent if the pH is to beraised, or, if the pH is to be lowered, with a suitable acid or acidreagent. The conversion of isoflavone conjugates to isoflavoneglucosides has been found to be base catalyzed, and so it is mostpreferred to utilize a high pH to achieve rapid conversion. The mostpreferred pH for conversion of the isoflavone conjugates to isoflavoneglucosides is a pH of about 9 to about 11.

The temperature range for conversion of the isoflavone conjugates toisoflavone glucosides in soy molasses is from about 2° C. to about 120°C. The temperature range at which the conversion readily occurs dependson the pH of the soy molasses material. The inventors have found thatthe conversion occurs easily at lower temperatures when the pH isrelatively high. For example, at a pH of about 11 the conversion occursrapidly and efficiently at a temperature range of about 50° C. to about50° C. At a pH of about 9 conversion occurs efficiently within atemperature range of about 45° C. to about 75° C. When the pH of the soymolasses is relatively low, the conversion occurs at highertemperatures. For example, at a pH of about 6, the conversion occurswithin a temperature range of about 80° C. to about 120° C. In apreferred embodiment, the conversion is effected at about 35° C. and apH of about 11. In another preferred embodiment, the conversion iseffected at a temperature of about 73° C. and a pH of about 9.

The time period required for conversion of isoflavone conjugates toisoflavone glucosides depends primarily upon the pH and temperaturerange utilized in the soy molasses. Such conversion times typicallyrange from about 15 minutes up to several hours or longer. Conversionoccurs more rapidly at a higher pH and at a higher temperature. At a pHof about 9, conversion is substantially complete in about 4 hours toabout 6 hours at 73° C. In a most preferred embodiment, the isoflavoneconjugates are converted to isoflavone glucosides in about 30 minutes toabout 1 hour, preferably about 45 minutes, at a pH of about 11 and at atemperature of about 35° C.

The conversion of the isoflavone conjugates to isoflavone glucosides isremarkably efficient, converting at least a majority, and preferablysubstantially all of the isoflavone conjugates present to isoflavoneglucosides. The term a "majority" refers to an extent of conversion ofat least about 50%. The term "substantially all" refers to an extent ofconversion of at least about 80%, and most preferably at least about90%.

Following the conversion of the isoflavone conjugates to isoflavoneglucosides, a cake of isoflavone glucoside enriched material may beseparated from the soy molasses material. The soy molasses material istreated at a pH and a temperature at which a majority of the isoflavoneglucosides will separate from the soy molasses material in a separationprocedure. In a preferred embodiment the soy molasses material ismaintained at a pH of about 3 to about 6.5, most preferably about 4.5,and at a temperature of about 0° C. to about 35° C., preferably about 0°C. to about 10° C., and most preferably about 4° C. to about 7° C.,during the separation process. The pH of the soy molasses material maybe adjusted with a suitable conventional acidic or basic reagent, ifnecessary.

The separation may be effected by conventional means for separatingsolids from a liquid. The isoflavone glucoside enriched cake ispreferably separated by centrifugation or filtration as described abovewith respect to separating an isoflavone enriched cake from a soymolasses material.

In yet another aspect of the invention, a cake of aglucone isoflavoneenriched material may be recovered from soy molasses. A soy molassesstarting material is obtained as described above, preferably dilutedwith water to a solids content of about 6% to about 13%. The soymolasses starting material is treated to convert the isoflavoneconjugates to isoflavone glucosides as described above.

An enzymatic conversion operation is then performed on the isoflavoneglucoside enriched soy molasses material by contacting a suitable enzymewith isoflavone glucosides in the soy molasses material at a suitable pHand temperature to convert the isoflavone glucosides to agluconeisoflavones. The two-step conversion process effectively convertssubstantially all of the isoflavone conjugates and isoflavone glucosidesin the soy molasses material to aglucone isoflavones, substantiallyincreasing the amount of aglucone isoflavones in the soy molassesmaterial.

The conversion of isoflavone glucosides to aglucone isoflavones has beenfound to be dependent on a variety of factors including the type ofenzymes present in the soy molasses material, distribution of enzymeconcentrations, activities of the enzymes, and the pH and temperature ofthe soy molasses material during the conversion. The enzymes required toeffect the conversion are enzymes capable of cleaving the glucosidiclinkage between the isoflavone moiety and the glucose molecule of theisoflavone glucosides. In a preferred embodiment, the enzymes aresaccharidase, or gluco-amylase enzymes capable of cleaving 1,4-glucosidebonds. The enzymes may be inherently present in the soy molassesmaterial, or may be commercially available enzymes which are added tothe soy molasses material. Inherently present enzymes are referred toherein as "residual" enzymes, and enzymes that are added to the soymolasses are referred to herein as "supplemental" enzymes.

Sufficient enzyme should be present in the soy molasses material toconvert at least a majority, and preferably substantially all, of theisoflavone glucosides to aglucone isoflavones. Generally, if theresidual enzymes in the soy molasses material are insufficient to effectthe conversion, supplemental enzymes should be added to the soy molassesmaterial. In a preferred embodiment, supplemental enzymes are added tothe soy molasses material regardless of whether sufficient residualenzymes are present in the soy molasses material since addition ofsupplemental enzymes dramatically decreases the time necessary to effectsubstantially complete conversion of the glucosides to aglucones. Ifsupplemental enzymes are added, the supplemental enzymes should be addedso that the total concentration of enzyme present is about 0. 1% toabout 10% by weight of the solids in the soy molasses material on a drybasis.

Supplemental enzymes are selected based on optimum activity at selectedpH and temperature conditions, and cost effectiveness. The supplementalenzymes are enzymes capable of cleaving the bond between the isoflavonemoiety and the glucose molecule of the isoflavone glucosides, such assacchariglucosides, such as saccharidase and gluco-amylase enzymecapable of cleaving 1,4-glucoside bonds. Preferred supplemental enzymesare commercially available alpha-and beta-glucosidase enzymes,beta-galactosidase enzymes, gluco-amylase enzymes, and pectinaseenzymes. Particularly preferred are enzymes such as Biopectinase 100L(which is preferably utilized at a pH range of from about 3 to about 6),Biopectinase 300L (optimum pH range from about 3 to about 6),Biopectinase OK 70L (optimum pH range from about 3 to about 6),Biolactase 30,000 (optimum pH range from about 3 to about 6) NeutralLactase (optimum pH range from about 6 to about 8), all of which areavailable from Quest International, 1833 57th Street, Post Office Box3917, Sarasota, Fla. 34243. Also especially preferred are Lactase F(which is preferably utilized at a pH range of from about 4 to about 6),and Lactase 50,000 (optimum pH range from about 4 to about 6), bothavailable from Amano International Enzyme Co., Inc., Post Office Box1000, Troy, Va. 22974. Other particularly preferred supplemental enzymesinclude G-Zyme G990 (optimum pH from about 4 to about 6) and EnzecoFungal Lactase Concentrate (optimum pH from about 4 to about 6)available from Enzyme Development Corporation, 2 Penn Plaza, Suite 2439,New York N.Y. 10121; Lactozyme 3000L (which preferably is utilized at apH range from about 6 to about 8), and Alpha-Gal 600L (which preferablyis utilized at a pH range of from about 4 to about 6.5), available fromNovo Nordisk Bioindustrials, Inc., 33 Turner Road, Danbury, Conn. 06813;Maxilact L2000 (which is preferably utilized at a pH range of from about4 to about 6), available from Gist Brocades Food Ingredients, Inc., Kingof Prussia, Pa., 19406; and Neutral Lactase (which is preferablyutilized at a pH range of from about 6 to about 8), available fromPfizer Food Science Group, 205 East 42nd Street, New York, N.Y. 10017.

The pH range for conversion of the isoflavone glucosides to agluconeisoflavones is from about 3 to about 9. The pH that is utilized dependsprimarily upon the type of enzyme used, and should be selectedaccordingly. The residual enzyme is active within a pH range of about 7to about 9, although it is believed that the pH of the soy molassesmaterial is lowered during the course of the conversion. Thesupplemental enzymes are active within an optimum pH range specified bythe manufacturer of the enzyme, as shown above for several specificenzymes. Typically the supplemental enzymes are active either in aneutral pH range from about 6 to about 8, or in an acidic pH range fromabout 3 to about 6.

The pH of the soy molasses material may be adjusted to a desired valuefor conducting the conversion of isoflavone glucosides to agluconeisoflavones. In most instances the pH is reduced from the relativelyhigh or basic pH required to convert the isoflavone conjugates toisoflavone glucosides by the addition of one or more suitable acids suchas acetic acid, sulfuric acid, phosphoric acid, hydrochloric acid, orany other suitable reagent.

The temperature range of the soy molasses material for the conversion ofglucosides to aglucones is from about 5° C. to about 75° C. Thetemperature significantly affects the activity of the enzymes, andtherefore, the rate of conversion. The supplemental enzymes may beactive above 70° C., for example Alpha-Gal 600L is active at 75° C.,however, it is preferred to conduct the conversion at lower temperaturesto avoid enzyme deactivation. In a preferred embodiment, the conversionis effected between about 35° C. and about 45° C.

The time required for conversion of the glucosides to aglucones dependsupon enzyme-related factors, particularly concentration, and thetemperature and pH of the system. In most instances it is possible toachieve substantially complete conversion within 24 hours, however, itis preferred that supplemental enzyme be added to dramatically increasethe rate of the reaction. The selected supplemental enzyme, enzymeconcentration, pH and temperature preferably cause substantiallycomplete conversion within about 2 hours, and most preferably withinabout 1 hour.

The conversion of the isoflavone glucosides to aglucone isoflavones isremarkably efficient, converting at least a majority, and preferablysubstantially all of the glucosides present to aglucones. The term "amajority" refers to an extent of conversion of at least about 50%. Theterm "substantially all" refers to an extent of conversion of at leastabout 80%, and most preferably at least about 90%.

Following the conversion of the isoflavone glucosides to agluconeisoflavones, a cake of aglucone isoflavone enriched material may beseparated from the soy molasses material. The soy molasses material istreated at a pH and a temperature at which a majority of the agluconeisoflavones will separate from the soy molasses material in a separationprocedure. Preferably, the soy molasses material is maintained at a pHof about 3 to about 6.5, most preferably about 4.5, and at a temperatureof about 0° C. to about 35° C., preferably about 0° C. to about 10° C.,and most preferably about 4° C. to about 7° C., during the separationprocess. The pH of the soy molasses material may be adjusted with asuitable conventional acidic or basic reagent, if necessary.

The separation may be effected by conventional means for separatingsolids from a liquid. The aglucone isoflavone enriched cake ispreferably separated by centrifugation or filtration as described abovewith respect to separating an isoflavone enriched cake from a soymolasses material.

An aglucone isoflavone enriched material may also be produced from anisoflavone enriched material recovered from soy molasses, where theprocess for recovering an isoflavone enriched material from soy molassesis described above. Water is added to the recovered cake of isoflavoneenriched material to form a slurry of the isoflavone enriched material.Preferably the slurry is diluted to about 6% to about 13% solids,although a higher solids content may be used. Isoflavone conjugates inthe slurry are then converted to isoflavone glucosides by treating theslurry under the same conditions described above with respect toconverting isoflavone conjugates to isoflavone glucosides in soymolasses. In particular, the slurry is treated at a pH of about 6 toabout 13.5, preferably about pH 9 to about pH 11, and a temperature ofabout 2° C. to about 1 20° C. for a period of about 15 minutes toseveral hours. Most preferably the slurry is treated at a pH of about 11and a temperature of about 5° C. to about 50° C., preferably about 35°C., for a period of about 30minutes to about 1 hour; or at a pH of about9 and a temperature of about 45° C. to about 75° C., preferably about73° C., for a period of about 4 hours to about 6 hours. If desired, anisoflavone glucoside enriched material may be separated from the slurryin a manner similar to the separation of an isoflavone enriched materialfrom soy molasses described above.

Isoflavone glucosides in the slurry are then converted to agluconeisoflavones under the same conditions described above with respect toconverting isoflavone glucosides to aglucone isoflavones in a soymolasses material. In particular, the isoflavone glucosides in theslurry are contacted with an enzyme capable of cleaving the glucosidiclinkage between the isoflavone moiety and the glucose molecule of theisoflavone glucosides under suitable pH and temperature conditions for aperiod of time sufficient to convert the isoflavone glucosides toaglucone isoflavones. Preferred enzymes, pH conditions, temperatures,and time periods are described above. An aglucone isoflavone enrichedmaterial may be separated from the slurry in a manner similar to theseparation of an isoflavone enriched material from soy molassesdescribed above.

An aglucone isoflavone enriched material may also be produced from anisoflavone glucoside enriched material recovered from a soy molassesmaterial, where the process for recovering an isoflavone glucosideenriched material from a soy molasses material is described above. Wateris added to the recovered cake of isoflavone glucoside enriched materialto form a slurry of the isoflavone glucoside enriched material.Preferably the slurry is diluted to about 6% to about 13% solids,although a higher solids content may be used. The isoflavone glucosidesin the slurry are converted to aglucone isoflavones in the same mannerdescribed above with respect to the isoflavone glucoside enriched slurryformed from an isoflavone enriched slurry. An aglucone isoflavoneenriched material may be separated from the slurry after the conversionin a manner similar to the separation of an isoflavone enriched materialfrom soy molasses described above.

EXPERIMENTAL

The present invention is illustrated in more detail by the followingexamples. The examples are intended to be illustrative, and should notbe interpreted as limiting or otherwise restricting the scope of theinvention in any way.

As noted above, soy materials, including soy molasses, include thegenistein, daidzein, and glycitein "families" of isoflavones havingcorresponding glucoside, conjugate, and aglucone members, where thegenistein family contains the conjugates 6"-OMal genistin, 6"-OAcgenistin, the glucoside genistin, and the aglucone genistein; thedaidzein family contains the conjugates 6"-OMal daidzin, 6"-OAc daidzin,the glucoside daidzin, and the aglucone daidzein; and the glyciteinfamily includes the conjugate 6"-OMal glycitin, the glucoside glycitin,and the aglucone glycitein. In the following examples the relativeconcentrations of isoflavones are measured either as a totalconcentration of an isoflavone family, or as individual percentages ofeach isoflavone in a family of isoflavones. For example, the totalconcentration of the genistein family of isoflavones is the sum of theconcentrations of 6"-OMal genistin, 6"-OAc genistin, genistin, andgenistein, and the percentage of each of the isoflavones in thegenistein family is determined relative to the other genistein familyisoflavones: % genistin +% 6"OMal genistin +% 6"OAc genistin +%genistein =100%.

EXAMPLE 1

In a first experiment, the recovery of an isoflavone enriched materialfrom soy molasses is examined at various concentrations of soy molasses.The total concentration of each isoflavone family is measured in a soymolasses sample having a selected concentration, in a cake separatedfrom the soy molasses sample according to the method of the invention,and in the liquid whey from which the cake is removed by the separationprocedure.

Soy molasses is analyzed for the total concentration of all forms ofisoflavones present. Samples of the soy molasses are diluted with waterto a solids content of 28% (1:2 dilution), 13.7% (1:4 dilution), and6.6% (1:8 dilution). All samples are pH adjusted to 4.5. The treatedsamples are then centrifuged at a rate of between 3000 rpm for 30minutes to separate and produce liquid whey and cake portions from thesamples. One set of samples is centrifuged at a temperature of 0.6° C.Samples having 28% and 13.7% soy molasses solids are centrifuged at atemperature of 60° C. for comparison with samples having the sameconcentration of soy molasses solids that are separated at 0.6° C. Theresulting liquid and cake portions of the samples are analyzed for thetotal concentration of all forms of isoflavones present.

Table 1 sets forth the concentrations of isoflavones in the various cakeand liquid fractions obtained from the previously described testing. Theindicated totals are the totals of all forms of the particularisoflavone including conjugates, glucoside, and aglucone forms expressedin mg of isoflavone per gram of cake or liquid fraction solids.

                                      TABLE 1                                     __________________________________________________________________________                Total    Total    Total                                                       Genistein (family)                                                                     Daidzein (family)                                                                      Glycitein (family)                              Sample      mg/g     mg/g     mg/g                                            __________________________________________________________________________    Soy Molasses                                                                  Starting Material                                                             Not separated                                                                             6.1      4.8      1.0                                             1:2 Dilution (28% solids)                                                     Whey separated at 0.6° C.                                                          2.8      3.0      0.6                                             1:2 Dilution                                                                  Cake separated at 0.6° C.                                                          16.9     10.9     1.9                                             1:2 Dilution                                                                  Whey separated at 60° C.                                                           3.8      4.0      0.8                                             1:4 Dilution (13.7% solids)                                                   Whey separated at 0.6° C.                                                          3.0      3.4      0.7                                             1:4 Dilution                                                                  Cake separated at 0.6° C.                                                          18.3     11.0     2.0                                             1:4 Dilution                                                                  Whey separated at 60° C.                                                           4.4      4.3      0.8                                             1:4 Dilution                                                                  Cake separated at 60° C.                                                           13.4     7.2      1.5                                             1:8 Dilution (6.6% solids)                                                    Whey separated at 0.6° C.                                                          4.3      4.5      0.9                                             1:8 Dilution                                                                  Cake separated at 0.6° C.                                                          20.1     10.2     2.1                                             __________________________________________________________________________

In all separated samples, the concentration of isoflavones issignificantly higher in the cake than in soy molasses starting materialand much higher than the concentration of isoflavones in the liquid wheyfraction solids. The samples separated at 0.6° C. contained a higherconcentration of isoflavones in the cake than corresponding samplesseparated at 60° C., which had higher concentrations of isoflavones inthe whey fraction solids.

EXAMPLE 2

In another experiment, the recovery of an isoflavone glucoside enrichedmaterial from soy molasses is examined. Isoflavone conjugates in the soymolasses are converted to isoflavone glucosides, and an isoflavoneglucoside enriched cake is separated from the soy molasses material. Theextent of conversion is determined by the quantitative decrease of thepercentage and concentration of malonate and acetate esters of anisoflavone family coupled with a corresponding quantitative increase ofthe percentage of the glucoside of the same isoflavone family.

Soy molasses starting material is analyzed for concentration ofindividual isoflavone compounds. Two samples of the soy molassesmaterial are made by diluting the soy molasses with water in thefollowing ratios: 1:4(100g of molasses +300g water); and 1:8(50g ofmolasses +350g water). The pH of the samples is adjusted to 11, and thetemperature of the samples is held at 35° C. for 30 minutes. The pH ofthe samples is then adjusted to 4.5 and the temperature is adjusted to4° C. The samples are centrifuged at 10,000 rpm at 4° C. to separate themolasses samples into a cake and a liquid whey. The whey and the cakeare analyzed for concentration of individual isoflavone compounds.

Table 2 illustrates the change in the proportions between the variousforms of isoflavones resulting from the conversion of isoflavoneconjugates to isoflavone glucosides as compared to the soy molassesstarting material. Isoflavone concentrations are indicated as parts permillion(ppm) within the sample, and as percentages of the total amountof the particular isoflavone (the total of the conjugate, glucoside, andaglucone forms) within the liquid or cake portion.

                                      TABLE 2                                     __________________________________________________________________________                  6"-  6"-           6"-  6"-            6"-                                    OMAL-                                                                              OAC-          OMAL-                                                                              OAC-           OMAL-                    Sample   Genistin                                                                           Genistin                                                                           Genistin                                                                           Genistein                                                                          Daidzin                                                                           Daidzin                                                                            Daidzin                                                                            Daidzein                                                                           Glycitin                                                                           Glycitin                                                                           Glycitein           __________________________________________________________________________    Soy Molasses                                                                  ppm      4678 1329 0    88   3533                                                                              928  210  84   500  105  360                 % isoflavone                                                                           77   22   0    1    74  20   4    2    52   11   37                  1:4 Dilution                                                                  Whey                                                                          ppm      2221 17   0    30   2652                                                                              179  29   21   341  28   0                   % isoflavone                                                                           98   1    0    1    92  6    1    1    92   8    0                   1:4 Dilution Cake                                                             ppm      28621                                                                              68   0    261  16133                                                                             192  0    232  1442 0    66                  % isoflavone                                                                           99   0    0    1    97  1    0    1    96   0    4                   1:8 Dilution                                                                  Whey                                                                          ppm      2852 24   0    36   3356                                                                              187  0    27   406  28   0                   % isoflavone                                                                           98   1    0    1    94  5    0    1    94   6    0                   1:8 Dilution Cake                                                             ppm      27517                                                                              101  0    272  12617                                                                             138  0    245  1146 0    0                   % isoflavone                                                                           99   0    0    1    97  1    0    2    100  0    0                   __________________________________________________________________________

In all samples subjected to conditions for conversion of isoflavoneconjugates to isoflavone glucosides the percentage of isoflavoneglucosides in both cake and liquid portions is significantly higher thanin the corresponding unconverted soy molasses sample, and the percentageof corresponding isoflavone conjugates in the samples is significantlylower, demonstrating that a large portion of isoflavone conjugates areconverted to their glucoside form. Furthermore, upon separation a largeproportion of the glucoside isoflavones are separated in the cake toform an isoflavone glucoside enriched material, as can be seen from therelative concentrations of the soy molasses starting material and thewhey and cake portions of each sample.

EXAMPLE 3

In another experiment, the conversion of isoflavones to agluconeisoflavones in soy molasses is examined. Isoflavone conjugates in thesoy molasses are converted to isoflavone glucosides, and the isoflavoneglucosides are then converted to aglucone isoflavones. The extent ofconversion of the isoflavone glucosides to aglucone isoflavones isdetermined by the quantitative decrease of the concentration of theglucoside of an isoflavone family coupled with a correspondingquantitative increase of the percentage of the aglucone of the sameisoflavone family.

Soy molasses starting material is diluted 1:4 with water and is analyzedfor concentration of individual isoflavone compounds. The pH of themolasses is then adjusted to 11. The soy molasses is held at roomtemperature for 1 hour to produce a glucoside enriched soy molassesmaterial. The glucoside enriched soy molasses material is analyzed forconcentration of individual isoflavone compounds. Four samples areprepared from the glucoside enriched soy molasses material after the pHof the material is adjusted to 4.5. Each sample is inoculated with anenzyme, where the following enzymes are added to the samples,respectively, at 10% by weight of the molasses solids in each sample:G-Zyme 990, Biopectinase 100 L, Lactase 50,000, and Alpha-Gal 600L. Thesamples are then treated at 50° C. for 6 hours to form an agluconeisoflavone enriched soy molasses material. The aglucone isoflavoneenriched soy molasses is then analyzed for isoflavone content.

Table 3 illustrates the distribution between the various forms ofisoflavones resulting from the previously described testing. Isoflavoneconcentrations are indicated as parts per million(ppm) within thesample, and as percentages of the total amount of the particularisoflavone (the total of the conjugate, glucoside, and aglucone forms).

                                      TABLE 3                                     __________________________________________________________________________                  6"-  6"-           6"-  6"-            6"-                                    OMAL-                                                                              OAC-          OMAL-                                                                              OAC-           OMAL-                    Sample   Genistin                                                                           Genistin                                                                           Genistin                                                                           Genistein                                                                          Daidzin                                                                           Daidzin                                                                            Daidzin                                                                            Daidzein                                                                           Glycitin                                                                           Glycitin                                                                           Glycitein           __________________________________________________________________________    Soy Molasses                                                                  ppm      4678 1329 0    88   3533                                                                              928  210  84   500  105  360                 % isoflavone                                                                           77   22   0    1    74  20   4    2    52   11   37                  Glucoside rich                                                                soy molasses                                                                  ppm      6763 0    0    104  4377                                                                              0    0    43   433  0    0                   % isoflavone                                                                           98   0    0    2    99  0    0    1    100  0    0                   G-Zyme 990, 10%                                                               ppm      3903 0    0    1993 840 0    82   2331 346  0    114                 % isoflavone                                                                           66   0    0    44   27  0    2    71   75   0    25                  Biopectinase                                                                  100L, 10%                                                                     ppm      2865 0    0    2919 541 0    94   2701 195  0    237                 % isoflavone                                                                           50   0    0    50   16       3    81   45   0    55                  Lactase 50,000,                                                               10%                                                                           ppm      0    0    0    4601 0   0    92   2875 0    0    366                 % isoflavone                                                                           0    0    0    100  0   0    3    97   0    0    100                 Alpha-Gal 600L,                                                               10%                                                                           ppm      28   0    0    4566 0   0    89   2882 0    0    356                 % isoflavone                                                                           1    0    0    99   0   0    3    97   0    0    100                 __________________________________________________________________________

The aglucone isoflavone content of the enzymatically treated samples issignificantly higher than the soy molasses and the glucoside enrichedsoy molasses material, indicating that the enzymatic treatment convertedsubstantial amounts of glucoside isoflavones to aglucone isoflavones.Selection of the proper enzyme, enzyme concentration, pH and temperaturefor the conversion permits conversion of substantially all of theisoflavone glucosides to aglucone isoflavones, as demonstrated by theisoflavone distribution in the Lactase 50,000 and Alpha-Gal 600Lsamples.

EXAMPLE 4

In a final experiment, the isoflavone content of an isoflavone enrichedmaterial, an isoflavone glucoside enriched material, and an agluconeisoflavone enriched material is examined and the distribution of theisoflavones in the materials is compared. Soy molasses is diluted to a1:4 ratio with water. A sample of the diluted soy molasses is adjustedto a pH of 4.5, is chilled to a temperature of 0.6° C. in an ice bathfor 30 minutes, and is centrifuged at a rate of 3000 rpm for 30 minutesto separate a cake of isoflavone enriched material. The remainingdiluted soy molasses is then adjusted to a pH of 11 with sodiumhydroxide and is treated at 50° C. for 1 hour to convert isoflavoneconjugates in the molasses to isoflavone glucosides. A sample of theglucoside enriched molasses is adjusted to a pH of 4.5,is chilled to atemperature of 0.6° C. in an icebath for 30 minutes, and is centrifugedat a rate of 3000 rpm for 30 minutes to separate a cake of isoflavoneglucoside enriched material. The remaining isoflavone glucoside enrichedsoy molasses material is adjusted to pH 4.5, and the enzyme G-Zyme 990is added to the material at a concentration of 2.6g enzyme/100g ofmolasses material. The enzyme and the isoflavone glucoside enriched soymolasses material are then treated at 50° C. for 18 to 20 hours toconvert the isoflavone glucosides to aglucone isoflavones. A sample ofthe aglucone isoflavone enriched soy molasses material is chilled to atemperature of 0.6° C. in an icebath for 30 minutes, and is centrifugedat a rate of 3000 rpm for 30 minutes to separate a cake of agluconeisoflavone enriched material. The recovered cakes of isoflavone enrichedmaterial, isoflavone glucoside enriched material, and agluconeisoflavone enriched material are then analyzed for isoflavone content.

Table 4 below shows the distribution of the isoflavones in the cakes ofisoflavone enriched material, isoflavone glucoside enriched material,and aglucone isoflavone enriched material. Isoflavone distribution isindicated as percentages of the total amount of the particularisoflavone (the total of the conjugate, glucoside, and aglucone forms).

                                      TABLE 4                                     __________________________________________________________________________                  6"-  6"-           6"-  6"-            6"-                                    OMAL-                                                                              OAC-          OMAL-                                                                              OAC-           OMAL-                    Sample   Genistin                                                                           Genistin                                                                           Genistin                                                                           Genistein                                                                          Daidzin                                                                           Daidzin                                                                            Daidzin                                                                            Daidzein                                                                           Glycitin                                                                           Glycitin                                                                           Glycitein           __________________________________________________________________________    Isoflavone rich                                                               material                                                                      % isoflavone                                                                           83   16   0    1    81  12   1    1    40   8    52                  Glucoside rich                                                                material                                                                      % isoflavone                                                                           99   0    0    1    99  0    0    1    95   0    5                   Aglucone rich                                                                 material                                                                      % isoflavone                                                                           3    0    0    97   0   0    0    100  54   18   28                  __________________________________________________________________________

The effectiveness of the conversion steps can be seen in the isoflavonedistribution of the materials. The isoflavone glucoside enrichedmaterial contains significantly higher amounts of isoflavone glucosidesthan the isoflavone enriched material and the aglucone isoflavonematerial, having an isoflavone content which is comprised of almostentirely isoflavone glucosides. The aglucone isoflavone enrichedmaterial contains significantly higher amounts of aglucone isoflavonesthan the isoflavone glucoside enriched material and the isoflavoneenriched material, having an isoflavone content which is comprised ofalmost entirely aglucone isoflavones.

In the above examples, all percentages indicated for 6"-OMal-genistin,6"-OAc-genistin, 6"-OMal-daidzin, 6"-OAc-daidzin, glycitin,6"-OMal-glycitin, and glycitein are calculated values. The following isa description of a method for quantifying isoflavones in soy products.The isoflavones are extracted from soy products by mixing 0.75 gram ofsample (spray dried or finely ground powder) with 50 ml of 80/20methanol/water solvent. The mixture is shaken for 2 hours at roomtemperature with an orbital shaker. After 2 hours, the remainingundissolved materials are removed by filtration through Whatman No. 42filter paper. Five ml of the filtrate are diluted with 4 ml of water and1 ml of methanol.

The extracted isoflavones are separated by HPLC (High Performance LiquidChromatography)using a Hewlett Packard C18 Hypersil reverse phasecolumn. The isoflavones are injected onto the column and eluted with asolvent gradient starting with 88% methanol, 10% water, and 2% glacialacetic acid and ending with 98% methanol and 2% glacial acetic acid. Ata flow rate of 0.4 ml/min, all the isoflavones - genistin,6"-0-acetylgenistin, 6"-0-malonylgenistin, genistein, daidzin,6"-0-acetyldaidzin, 6"-0-malonyldaidzin, daidzin, glycitin and itsderivatives and glycitein - are clearly resolved. Peak detection is byUV absorbence at 260 mm. Identification of the peaks was performed byHPLC-mass spectrometer.

Quantification is achieved by using pure standards (genistin, genistein,daidzin and daidzein) obtained from Indofine Chemical Company,Sommerville, N.J. Response factors (integrated area/concentration) arecalculated for each of the above compounds and are used to quantitateunknown samples. For the conjugated forms for which no pure standardsare available, response factors are assumed to be that of the parentmolecule but corrected for molecular weight difference. The responsefactor for glycitin is assumed to be that for genistin corrected formolecular weight difference. This method provides the quantities of eachindividual isoflavone. For convenience, total genistein, total daidzeinand total glycitein can be calculated, and represent the aggregateweight of these compounds if all the conjugated forms are converted totheir respective unconjugated forms. These totals can also be measureddirectly by a method using acid hydrolysis to convert the conjugatedforms.

The foregoing are merely preferred embodiments of the invention. Variouschanges and alterations can be made without departing from the spiritand broader aspects thereof as set forth in the appended claims, whichare to be interpreted in accordance with the principals of patent lawincluding the Doctrine of Equivalents.

What is claimed is:
 1. A process for obtaining an isoflavone glucosidematerial from soy molasses containing isoflavone conjugates,comprising:providing a soy molasses material containing isoflavoneconjugates: treating said soy molasses material at a pH and atemperature effective to convert isoflavone conjugates to isoflavoneglucosides for a time period sufficient to convert isoflavone conjugatesto isoflavone glucosides; and separating an isoflavone glucosideenriched material from said soy molasses.
 2. The process as set forth inclaim 1 wherein said slurry is treated at a pH of about 6 to about 13.5and at a temperature of about 2° C. to about 120° C. to convert saidisoflavone conjugates to isoflavone glucosides.
 3. The process as setforth in claim 2 wherein said soy molasses is treated at a pH of about 9to about 11 and at a temperature of about 5° C. to about 75° C. for aperiod of at least about 15 minutes to convert said isoflavoneconjugates to isoflavone glucosides.
 4. The process as set forth inclaim 3 wherein said soy molasses is treated at a pH of about 11 and atemperature of about 5° C. to about 50° C. for a period of about 15minutes to about 1 hour to convert said isoflavone conjugates toisoflavone glucosides.
 5. The process as set forth in claim 4 whereinsaid soy molasses is treated at a pH of about 11 and at a temperature ofabout 35° C. for about 30 minutes to about 1 hour to convert saidisoflavone conjugates to isoflavone glucosides.
 6. The process as setforth in claim 3 wherein said soy molasses is treated at a pH of about 9and a temperature of about 45° C. to about 75° C. for about 4 hours toabout 6 hours to convert said isoflavone conjugates to isoflavoneglucosides.
 7. The process as set forth in claim 1 wherein saidisoflavone glucoside enriched material is separated from said soymolasses at a pH of about 3 to about 6.5 and a temperature of about 0°C. to about 35° C.
 8. The process as set forth in claim 7 wherein saidisoflavone glucoside enriched material is separated from said soymolasses material at a temperature of about 0° C. to about 10° C.
 9. Theprocess as set forth in claim 7 wherein said isoflavone glucosideenriched material is separated from said soy molasses material at a pHof about 4.5.
 10. The process as set forth in claim 7 wherein the stepof separating said isoflavone glucoside enriched material from said soymolasses comprises centrifugation.
 11. The process as set forth in claim7 wherein the step of separating said isoflavone glucoside enrichedmaterial from said soy molasses material comprises filtration.
 12. Theprocess of claim 1 further comprising:forming an aqueous slurry of saidseparated isoflavone glucoside enriched material; and contacting anenzyme with said isoflavone glucosides in said slurry at a pH and atemperature for a time sufficient to convert said isoflavone glucosidesto aglucone isoflavones to form an aglucone isoflavone enriched slurry.13. The process as set forth in claim 12 wherein said enzyme iscontacted with said isoflavone glucosides in said slurry at a pH ofabout 3 to about 9 and at a temperature of about 5° C. to about 75° C.for at least about 1 hour.
 14. The process as set forth in claim 12wherein contacting an enzyme with said isoflavone glucosides in saidslurry comprises adding an effective amount of a supplemental enzyme tosaid slurry.
 15. The process as set forth in claim 14 wherein saidsupplemental enzyme comprises a saccharidase enzyme capable of cleaving1,4-glucoside bonds.
 16. The process of claim 12 further comprising,after converting said isoflavone glucosides to aglucone isoflavones,separating an aglucone isoflavone enriched material from said slurry.17. The process as set forth in claim 16 wherein said agluconeisoflavone enriched material is separated from said slurry at a pH ofabout 3 to about 6.5 and at a temperature of about 0° C. to about 35° C.18. The process as set forth in claim 17 wherein said agluconeisoflavone enriched material is separated from said slurry at atemperature of about 0° C. to about 10° C.
 19. The isoflavone glucosideenriched material produced by the process set forth in claim
 1. 20. Theaglucone isoflavone enriched material produced by the process set forthin claim 16.